Structure of aspartoacylase, the brain enzyme impaired in Canavan disease

Abstract

Aspartoacylase catalyzes hydrolysis of N-acetyl-L-aspartate to aspartate and acetate in the vertebrate brain. Deficiency in this activity leads to spongiform degeneration of the white matter of the brain and is the established cause of Canavan disease, a fatal progressive leukodystrophy affecting young children. We present crystal structures of recombinant human and rat aspartoacylase refined to 2.8-and 1.8-Å resolution, respectively. The structures revealed that the N-terminal domain of aspartoacylase adopts a protein fold similar to that of zinc-dependent hydrolases related to carboxypeptidases A. The catalytic site of aspartoacylase shows close structural similarity to those of carboxypeptidases despite only 10 -13% sequence identity between these proteins. About 100 C-terminal residues of aspartoacylase form a globular domain with a two-stranded ␤-sheet linker that wraps around the N-terminal domain. The long channel leading to the active site is formed by the interface of the N-and C-terminal domains. The C-terminal domain is positioned in a way that prevents productive binding of polypetides in the active site. The structures revealed that residues 158 -164 may undergo a conformational change that results in opening and partial closing of the channel entrance. We hypothesize that the catalytic mechanism of aspartoacylase is closely analogous to that of carboxypeptidases. We identify residues involved in zinc coordination, and propose which residues may be involved in substrate binding and catalysis. The structures also provide a structural framework necessary for understanding the deleterious effects of many missense mutations of human aspartoacylase.N-acetyl-L-aspartate ͉ x-ray structure ͉ zinc-dependent hydrolase

Mutational analysis of aspartoacylase: Implications for Canavan Disease

“…Finally, the distance between the sulfur atoms of Cys124 and Cys152 is 5. 5 Å (Bitto et al 2007), which means they are close enough for the formation of a disulfide bridge. Future studies would benefit from the proposed homology model and proposed catalytic mechanism, as well as the available crystal structure for drug design with the goal of restoring ASPA activity in CD patients with catalytically deficient ASPA enzyme (Qiao et al 2006).…”

“…During the preparation of this manuscript, a report was published which confirms our proposal that ASPA contains a single zinc atom (Le Coq et al 2006). When this manuscript was in the process of review, the X-ray crystal structures of rat and human ASPA were published (Bitto et al 2007). Upon comparing our homology model of the active and binding sites of ASPA with those of the published crystal structure, the root-mean-square deviations between C α atoms is 1.1 Ǻ.…”

Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue,

“…However, the sequence of human aspartoacylase is only ~10% identical with that of bovine carboxypeptidase A (CPA) (8). A structural comparison of rat aspartoacylase with CPA found that the C-terminal domain of aspartoacylase is not present in carboxypeptidase, while the Nterminal domain of CPA is approximately 60 residues longer than the corresponding domain of aspartoacylase (10).…”

“…This classification is supported by recent work in which aspartoacylase was shown to bind one zinc ion per monomer (9), consistent with its assignment to the zinc carboxypeptidase family. Recently, the crystal structures of the rat form and the human form of aspartoacylase have been determined at 1.8 and 2.8 Å resolution, respectively, by the Center for Eukaryotic Structural Genomics (10). Both of these structures are the apo form of the enzyme that was crystallized in the absence of either substrates or products.…”

“…These structures confirm the presence of zinc in the enzyme, as well as the identity of the zinc binding ligands which had been proposed previously. In addition, the ligands that are potentially involved in substrate binding have been suggested through a comparison of the structure of aspartoacylase with those of the carboxypeptidases (10). However, the sequence of human aspartoacylase is only ~10% identical with that of bovine carboxypeptidase A (CPA) (8).…”